Raman tag

ABSTRACT

A method of forming a probe, wherein the method includes converting cholenic acid into a compound with a terminal alkyne group, wherein the converting the cholenic acid comprises using a sequence, wherein the sequence comprises synthesizing a THP-protection group, LiAlH4 reduction, Dess-Martin oxidation, and Seyferth-Gilbert-Bestmann homologation.

CROSS REFERENCE TO RELATED APPLICATIONS

The Present U.S. patent application is a continuation of U.S. patentapplication Ser. No. 15/634,567, filed Jun. 27, 2017, is a continuationof U.S. patent application Ser. No. 14/850,949, filed Sep. 10, 2015,which is related to and claims the priority benefit of U.S. ProvisionalPatent Application Ser. No. 62/048,484, filed Sep. 10, 2014, thecontents of which are hereby incorporated by reference in their entiretyinto this disclosure.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under CA182608 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

The present disclosure generally relates to tags for imaging moleculesusing Raman spectroscopy, and in particular to a method and compositionthat uses cholesterol mimics to track the location and movement ofcholesterol.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

An important component of cellular membrane, cholesterol controlsphysical properties of the membrane and contributes to specific membranestructures such as lipid rafts. Inside cells, cholesterol plays animportant role in various signaling pathways and serves as the precursorfor signaling molecules, and modifies specific proteins, such ashedgehog, to control protein trafficking and activity. The distributionof cholesterol in a living cell is highly regulated. Intracellularcholesterol is stored in lipid droplets (LDs) in the form of cholesterylester to avoid the toxicity caused by free cholesterol. Dysregulation ofcholesterol metabolism and/or trafficking has been linked to variousdiseases, including atherosclerosis, Niemann-Pick type C (NP-C) disease,and various cancers.

Intracellular cholesterol transport and metabolism have been studiedextensively using various reporter molecules, including cholesterolbinding molecules and cholesterol analogs. Cholesterol bindingmolecules, such as cholesterol oxidase, filipin, and perfringolysin Oderivatives, are commonly used to study steady-state distribution ofcholesterol in fixed cells and tissues. Fluorescent cholesterol,including intrinsic fluorescent sterols (DHE for dehydroergosterol) andfluorophore-tagged analogs (NBD-cholesterol and BODIPY-cholesterol) arewidely used in vitro and in vivo. Radiolabeled cholesterol or itsprecursors are used in biochemical studies of metabolism and traffickingof cholesterol.

These current cholesterol assays have limitations. Cholesterol oxidaseis commonly used in fluorometric or colorimetric assays to quantifytotal cholesterol in homogenized cells. Radiolabeled cholesterol has tobe used in combination with separation methods to determineintracellular cholesterol distribution indirectly. For imaging purpose,filipin is the most commonly used molecule for visualizing distributionof free cholesterol, but it is only applicable to fixed cells or tissueswith moderate specificity because filipin also labels other lipids.Fluorescent BODIPY-cholesterol is known to cause perturbations due tobulkiness of the fluorophore. DHE has the closest structure ascholesterol, but its fluorescence undergoes rapid photo-bleaching, whichimpedes real-time observation of cholesterol trafficking. There exists aneed for new technologies that allow for real time imaging ofcholesterol transport and low toxicity in live cells.

SUMMARY OF THE INVENTION

The present invention provides novel Raman tags for exploring membraneinteractions in cells. The tags comprise a phenyl diyne probe where inthe dyine is capped with the phenyl group. Methods for using the tagsare also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Design and synthesis of tagged cholesterol probes. Reagents andconditions: a) DHP (5.0 equiv), p-TsOH (0.2 equiv), THF, RT, 91%; b)LiAlH₄ (3.0 equiv), THF, 0° C. RT, 98%; c) DMP (3.0 equiv), NaHCO₃ (3.0equiv), CH₂Cl₂, 0° C., 86%; d) dimethyl (1-diazo-2-oxopropyl)phosphonate(Bestmann reagent, 2.4 equiv), K₂CO₃ (4.0 equiv), THF/MeOH, RT, 99%; e)p-TsOH (1.0 equiv), THF/MeOH, RT, 84%; f) Iodobenzene (1.02 equiv),PdCl₂(PPh₃)₂ (0.05 equiv), CuI (0.05 equiv), TEA, RT; then TsOH (1.0equiv), THF/MeOH, RT, 77%; g) CuI (0.1 equiv), K₂CO₃ (2.0 equiv),P(o-Tol)₃ (0.2 equiv), phenyl bromoacetylene (1.3 equiv), EtOH, 100° C.,51%; h) TsOH (1.0 equiv), THF/MeOH, RT, 95%; i) MsCl (3.0 equiv), TEA(3.0 equiv), CH₂Cl₁, 0° C. RT, 75%; j) KCN (2.0 equiv), DMSO, 90° C.,76%; k) TsOH (1.0 equiv), THF/MeOH, RT, 74%. DHP=3,4-Dihydro-2H-pyran,DMP=Dess-Martin Periodinane, p-TsOH=p-Toluenesulfonic acid,TEA=triethylamine, P(o-Tol)₃=tri(o-tolyl)phosphine, MsCl=methanesulfonylchloride. CN: cyano; A: alkyne; PhA: phenyl-alkyne; PhDY: phenyl-diyne;Chol: cholesterol.

FIG. 2. Raman spectral analysis of tagged cholesterol and SRS detectionof PhDY-Chol. (a) Raman spectra of 50 mM tagged cholesterols incyclohexanone (solvent). Spectral intensity was normalized by C═Ovibration band at 1,714 cm⁻¹. Spectral acquisition time: 10 s. (b) Plotof relative intensity of Raman tags versus solvent and Raman shifts oftagged cholesterols. Based on the molar concentration of the molecules(50 mM) and the solvent (9.7 M), the Raman intensities of C≡C fromA-Chol, PhA-Chol, and PhDY-Chol are 11 times, 16 times, and 122 timeshigher than the C═O band from the solvent, respectively. CN: cyano; A:alkyne; PhA: phenyl-alkyne; PhDY: phenyl-diyne; Chol: cholesterol. (c)SRS contrast versus concentration plot of PhDY-Chol solutions. 13%contrast was reached at 313 μM and 4% contrast was reached at 156 μM.Image acquisition speed: 200 s per pixel. Data represents the mean±SEMin 3 measurements. R²=0.996. (d) SRS contrast versus concentration plotof PhDY-Chol solutions using chirped femtosecond lasers with spectralfocusing approach. 14% contrast was reached at 31 μM. Image acquisitionspeed: 200 s per pixel. Data represents the mean±SEM in 3 measurements.R²=0.980. Contrast was defined as (S−B)/B. S: SRS signal; B: background.

FIG. 3. Cell-viability assays showing that phenyl group reducescytotoxicity of the probes. MTT Cell-viability assays show that A-Cholis toxic to the cells, but phenyl group prevents the cytotoxicity. CHOcells were incubated with each probe in various concentrations for 48 hbefore MTT cell-viability assays were conducted. Error bars representstandard error of the mean (SEM). n>3, *: p<0.05; **: p<0.005; ***:p<0.0005. A: alkyne; PhA: phenyl-alkyne; PhDY: phenyl-diyne; Chol:cholesterol.

FIG. 4. SRS images of PhDY-Chol in live CHO cells and blockage ofPhDY-Chol storage into LDs via ACAT-1 inhibition. (a) SRS images of liveCHO cells treated with PhDY-Chol (50 μM) for 16 h. C≡C vibrational modeat 2,252 cm⁻¹ was used for PhDY-Chol, and C—H vibrational mode at 2,885cm⁻¹ was used for C—H-rich lipid structures. Lasers were also tuned awayto 2,099 cm⁻¹ to show specificity of PhDY-Chol signal inside the cells.PhDY-Chol was found to accumulate in LDs (arrows). Image acquisitionspeed: 10 μs per pixel for 512×512 pixels. Scalar bar: 10 μm. (b)Schematic graph showing the hypothesis of PhDY-Chol metabolism insidethe cells. ACAT-1: Acyl-CoA:cholesterol acyltransferase. (c) SRS imagesof PhDY-Chol in CHO cells and ACAT-1 inhibited CHO cells by avasimibetreatment. As shown in circles, PhDY-Chol was stored in LDs in CHOcells, but not in avasimibe treated CHO cells. Image acquisition speed:100 μs per pixel for 400×400 pixels. Scalar bar: 10 μm. Intensity barsin a and c show the ΔI/I of the SRS image. (d) Quantification ofPhDY-rich and BODIPY-rich LDs in CHO cells before and after ACAT-1inhibition. The number of the LDs was normalized by the control group(n=7). Error bars represent standard deviation. *: p<0.05. (e) TPEFimages of BODIPY-cholesterol and SRS images C—H-rich structures in CHOcells and ACAT-1 inhibited CHO cells. As shown in circles,BODIPY-cholesterol showed no difference between the two groups. Scalarbar: 10 μm.

FIG. 5. Restored cholesterol transport in M12 cells treated with HPβCD.TPEF images of filipin and SRS images PhDY-Chol in (a) PhDY-Chol-loadedM12 cells, and (b) the same cells treated with HPβCD (500 μM) for 30 h.Arrows indicate PhDY-rich area labeled by filipin before treatment(non-esterified PhDY-Chol), and arrow heads indicate PhDY-rich area notlabeled by filipin after treatment (esterified PhDY-Chol). Greenintensity bar shows the ΔI/I value of the SRS image; red intensity barrepresents the relative intensity of fluorescence. Image acquisitionspeed: 100 μs per pixel for 400×400 pixels. Scalar bar: 10 μm. (c)Quantification of PhDY-rich area in the cells before and after HPβCDtreatment (n=7). Error bars represent standard deviation. *: p<0.05. (d)TPEF images of BODIPY and SRS images of PhDY-Chol in M12 cells treatedwith or without HPβCD (500 μM) for 30 h. Arrow heads indicate LDswithout PhDY-Chol before treatment, and arrows indicate LDs withPhDY-Chol after treatment. Green intensity bar shows the ΔI/I value ofthe SRS image; red intensity bar represents the relative intensity offluorescence. Image acquisition speed: 100 μs per pixel for 400×400pixels. Scalar bar: 10 μm. (e) Quantification of PhDY-rich LDs in thecells before and after HPβCD treatment (n=7). Error bars representstandard deviation. **: p<0.005.

FIG. 6. SRS imaging of PhDY-Chol visualizes compartments of cholesterolstorage in live C. elegans. (a) SRS images of live wildtype and ChUP-1deleted C. elegans fed with PhDY-Chol (500 μM) for 3 days. Arrowsindicate PhDY-rich particles in the intestine. Image acquisition speed:10 s per pixel for 400×400 pixels. Scalar bar: 10 m. (b) TPEF and SRSimages of live hjIs9 [ges-1p::glo-1::GFP+unc-119(+)] worm fed withPhDY-Chol (500 μM) for 3 days. Arrows indicate the PhDY-rich particlesin LROs. Image acquisition speed: 10 μs per pixel for 400×400 pixels.Scalar bar: 10 μm.

FIG. 7. Theoretical Raman intensities of the C≡C stretching mode invarious tags. The total molecular polarizability is broken down in termsof the value of the polarizability corresponding to each bond in themolecule. The Raman scattering cross section arises from thepolarizability caused by conjugation of the it-electrons of the alkyneand the phenyl groups. The π-orbitals possess large polarizabilitytensors, such that the polarizability of the triple bond is mainlydetermined by the polarizability of ir-orbitals. (a) Depiction ofsymmetry-localized π-orbitals in C₄H—C₆H₅(C≡C—C≡C-Ph). The distributedpolarizabilities corresponding to the localized orbitals on C≡C andphenyl ring are shown in Supplementary Table 1, such that thepolarizabilities of orbitals 1 and 2 determine the total polarizabilityof the left C≡C bond, polarizabilities of orbitals 3 and 4 sum up to thepolarizability of the middle C≡C bond. Additionally, the closest toalkyne part of the ring (orbital 5) exhibits significant change inpolarizability along triple-bond stretching vibration, and thereforepolarizabilities corresponding to this orbital are included in the totalcount of polarizability of the triple-bond system. Distributedpolarizabilities are calculated using GAMESS electronic structurepackage¹. (b) Comparison of C≡C stretching Raman intensities in varioustags computed using the Q-Chem quantum chemistry software². Intensitieswere normalized to the highest intensity.

FIG. 8. Linear correlation between PhDY-Chol concentration andmodulation depth. Concentration of PhDY-Chol and modulation depth (ΔI/I)show linear correlation, which can be expressed as: y=0.85x−5.77(R²=0.98). Inset is an SRS image of 10 mM PhDY-Chol in cyclohexanone.Data acquisition speed: 200 μs per pixel. Error bars represent standarderrors.

FIG. 9. Phenyl group prevented cytotoxicity of the probe molecules.Propidium iodide staining show that alkyne-cholesterol induces apoptosisand necrosis. Transmission images show reduced cell number inalkyne-cholesterol treated CHO cells. Phenyl group prevented thecytotoxic effect. A-Chol: alkyne cholesterol; PhA-Chol: phenyl-alkynecholesterol; PhDY-Chol: phenyl-diyne cholesterol; PI: propidium iodide.Scalar bar: 50 μm.

FIG. 10. PhDY-Chol is incorporated into cellular membrane. (a) SRSimages of live CHO cells. Cells were trypsinized and suspended inmedium. PhDY-Chol was seen in plasma membrane and intracellularstructures. Data acquisition time: 10 μs per pixel for 800×800 pixels.Scalar bar: 10 μm. (b) TPEF image of filipin-labeled CHO cells. Arrowindicates the point used for Raman spectral analysis. Red intensity barrepresents the relative intensity of fluorescence. Scalar bar: 10 μm.(c) Raman spectrum of filipin-labeled cell membrane acquired on the sameTPEF microscope. The bands for filipin, protein (amide I), and C≡Cvibrational mode are indicated by black arrows. Spectrum acquisitiontime: 30 s.

FIG. 11. TPEF imaging and Raman spectral analysis to confirm thatPhDY-Chol is stored into LDs. (a) TPEF image of BODIPY-labeled CHOcells. Arrow indicates the point used for Raman spectral analysis. Redintensity bar represents the relative intensity of fluorescence. Scalarbar: 10 m. (b) Raman spectrum of BODIPY-labeled LDs acquired on the sameTPEF microscope. The bands for cholesterol ring, BODIPY, C≡C, and C—Hvibrational modes are indicated by black arrows. Spectrum acquisitiontime: 30 s.

FIG. 12. ACAT-1 inhibition blocks PhDY-CHOL storage into LDs. (a) SRSimages of ACAT-1 knocked down CHO cells. No overlap was observed betweenPhDY-rich particles and LDs. Intensity bar shows the ΔI/I value of theSRS image. Image acquisition speed: 10 s per pixel for 400×400 pixels.Scalar bar: 10 μm. (b) SRS images of PhDY-Chol and TPEF images ofLysoTracker-stained organelles in CHO cells and ACAT-1 inhibited CHOcells by avasimibe treatment. PhDY-Chol was overlapped with LDs but notwith LysoTracker-stained organelles in control CHO cells. Afteravasimibe treatment, PhDY-Chol was not overlapped with LDs but withLysoTracker-stained organelles (circles). Image acquisition speed: 10 sper pixel for 400×400 pixels. Scalar bar: 10 m.

FIG. 13. PhDY-Chol reflects lysosomal cholesterol accumulation in M12cells. (a) TPEF image of filipin-labeled M12 cells. Arrow indicates thepoint used for Raman spectral analysis. Red intensity bar represents therelative intensity of fluorescence. Image acquisition speed: 10 s perpixel for 400×400 pixels. Scalar bar: 10 m. (b) Raman spectrum of thefilipin-labeled organelle acquired on the same TPEF microscope. Thebands for cholesterol ring, filipin, protein (amide I), and C≡Cvibrational mode are indicated by black arrows. Spectrum acquisitiontime: 30 s. (c) SRS image of PhDY-Chol and TPEF image ofLysoTracker-stained organelles in M12 cells. All PhDY-CHOL was foundinside lysosomes. Arrows representatively indicate that PhDY-Chol isaccumulated in lysosomes. Intensity bar shows the ΔI/I value of theimage. Image acquisition speed: 10 μs per pixel for 400×400 pixels.Scalar bar: 10 μm.

FIG. 14. The photostability of PhDY-Chol and the photo-bleaching ofBODIPY-Chol. The SRS images of 50 mM PhDY-Chol solution and TPEF imagesof 50 mM BODIPY-Chol solution were acquired continuously for 150 s (oneacquisition every 30 s). No significant change of the SRS signal wasobserved for PhDY-Chol, and a rapid photo-bleaching was observed forBODIPY-Chol.

FIG. 15. ¹H NMR of S1 (500 MHz, CDCl₃)

FIG. 16. ¹³C NMR of S1 (100 MHz, CDCl₃)

FIG. 17. ¹H NMR of S2 (400 MHz, CDCl₃)

FIG. 18. ¹³C NMR of S2 (100 MHz, CDCl₃)

FIG. 19. ¹H NMR of S3 (400 MHz, CDCl₃)

FIG. 20. ¹³C NMR of S3 (100 MHz, CDCl₃)

FIG. 21. ¹H NMR of 4 (500 MHz, CDCl₃)

FIG. 22. ¹³C NMR of 4 (125 MHz, CDCl₃)

FIG. 23. ¹H NMR of 5 (400 MHz, CDCl₃)

FIG. 24. ¹³C NMR of 5 (100 MHz, CDCl₃)

FIG. 25. ¹H NMR of S4 (400 MHz, CDCl₃)

FIG. 26. ¹³C NMR of S4 (125 MHz, CDCl₃)

FIG. 27. ¹H NMR of S5 (500 MHz, CDCl₃)

FIG. 28. ¹³C NMR of S5 (125 MHz, CDCl₃)

FIG. 29. ¹H NMR of 8 (400 MHz, CDCl₃)

FIG. 30. ¹³C NMR of 8 (100 MHz, CDCl₃)

FIG. 31. ¹H NMR of S6 (400 MHz, CDCl₃)

FIG. 32. ¹³C NMR of S6 (100 MHz, CDCl₃)

FIG. 33. ¹H NMR of 6 (400 MHz, CDCl₃)

FIG. 34. ¹³C NMR of 6 (100 MHz, CDCl₃)

FIG. 35. ¹H NMR of S7 (400 MHz, CDCl₃)

FIG. 36. ¹³C NMR of S7 (100 MHz, CDCl₃)

FIG. 37. ¹H NMR of 7 (400 MHz, CDCl₃)

FIG. 38. ¹³C NMR of 7 (100 MHz, CDCl₃)

FIG. 39. ¹H NMR of S8 (500 MHz, CDCl₃)

FIG. 40. ¹³C NMR of S8 (100 MHz, CDCl₃)

FIG. 41. ¹H NMR of 8 (400 MHz, CDCl₃)

FIG. 42. ¹³C NMR of 8 (125 MHz, CDCl₃)

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended. The terms “I,”“we,” “our” and the like throughout the Detailed Description, do notrefer to any specific individual or group of individuals.

A novel composition and a method that allows bio-orthogonal imaging ofcholesterol esterification, storage, and trafficking inside living cellsand vital organisms. By rational design and chemical synthesis, weprepared a probe molecule, phenyl-diyne cholesterol (PhDY-Chol), whichgives a 2,254 cm⁻¹ Raman peak that is 122 times stronger than theendogenous C═O stretching band. Compared to alkyne-cholesterol mimic ofwhich the IC₅₀ is 16 μM, the phenyl-diyne group is biologically inertand did not cause cytotoxicity after 16 h incubation at 50 μM. In liveChinese hamster ovary (CHO) cells, SRS imaging showed incorporation intoplasma membrane, esterification of PhDY-Chol by acyl-CoA: cholesterolacyltransferase 1 (ACAT-1), and storage in lipid droplets (LDs). In acellular model of NP-C disease, PhDY-Chol is selectively accumulated inlysosomes and is esterified and relocated to LDs after treatment with acholesterol-mobilization drug. In live C. elegans, SRS imaging ofPhDY-Chol revealed a previously unnoticed compartment of cholesterolstorage, regulated by the cholesterol uptake protein ChUP-1. Thesestudies herald the potential of the method for unveiling intracellularcholesterol trafficking mechanisms and highly efficient screening ofdrugs that target cholesterol metabolism.

The following describes compositions, methods of synthesis, and methodsof use of these compositions in the study of cholesterol. Thesecompositions may be used to study cholesterol localization and movementwithin live cells. The cholesterol mimics may be used to study andunderstand different changes in metabolism and organization by trackingthe changes in localization and modification of cholesterol. Thecholesterol mimics may be used to generate assays for screening leadcompounds or treatments to prevent or treat a metabolic irregularity ordisease such as cancer. In other aspects the cholesterol mimic may beused to study the lipid droplets with in a given cell, healthy or nothealthy, to provide an analysis. The cholesterol mimes may be used as atarget in a drug delivery system, where the drug targets the mimic in acertain unmodified or modified state, specifically. The cholesterolmimics may be used to provide a diagnosis or monitor the health of apatient's cells before or during treatment for a disease. Thecholesterol mimics may be used in vitro or in vivo (i.e. cells andtissues, whole organs, or live animals or humans), and may be analyzedin live or fixed cells.

The cholesterol mimics may be provided for use in a powder or crystalform, or suspended in liquid. The cholesterol mimics may be supplied byseveral delivery means including but not limited to orally,intravenously, injection, inhalation, catheter, dermal absorption, oringestion.

In general, a cholesterol mimic is generated by replacing the aliphaticchain of in cholesterol with a group that changes the Raman spectraproduced during Raman spectroscopy. Ideally, a peak is generated fromthis molecule that is differentiated from other signals produced by acell or tissue. One aspect, is to replace the aliphatic chain ofcholesterol with a group that will generate a peak between 1,800 and2,800 cm⁻¹ in a Raman scatter. One way to produce a signal in thisregion is to add a C≡C moiety to cholesterol, as shown in FIG. 1. Incertain aspects a C≡C bond may be added one, two, three, four, five, orsix times to increase signal. In certain aspects there are three C≡Cbonds included in the cholesterol mimic.

In certain aspects the PhDY-Chol mimic probe was generated by replacingthe aliphatic chain in cholesterol with phenyl-diyne. The followingchemical structures illustrate some of the mimics:

In certain aspects of the technology, the mimic is viewed by Ramanspectroscopy. Those of skill in the art will understand the set up andlaser sources to use given the chemical nature of the cholesterol mimic.For illustrative purposes only, one may look to the contents of theFigures, Brief Descriptions of the Figures and Detailed Description ofthis Application, to understand one example of a setup for performingRaman spectroscopy using the disclosed cholesterol mimics.

Cholesterol mimics were synthesized and a few specific embodiments willnow be described. Referring now to FIG. 1, alkyne cholesterol (A-Chol,5), phenyl-alkyne cholesterol (PhA-Chol, 6), phenyl-diyne cholesterol(PhDY-Chol, 7), and cyano cholesterol (CN-Chol, 8). Initially, synthesiscommenced with cholenic acid 3. Using a sequence of THP-protection,LiAlH₄ reduction, Dess-Martin oxidation and Seyferth-Gilbert-Bestmannhomologation, cholenic acid 3 was converted to compound 4 intermediatewith a terminal alkyne group. Removal of the THP-protecting group gaveprobe A-Chol 5. PhA-Chol 6 and PhDY-Chol 7 were prepared from compound 4intermediate via a palladium-catalyzed Sonogashira reaction and acopper-catalyzed Cadiot-Chodkiewicz reaction, respectively, followed byacidic removal of THP group. Additionally, CN-Chol 8 was prepared fromcholenic acid 3 via standard transformations.

The following are specific examples and embodiments, and not meant to belimiting in any way.

Design and Synthesis of Composition

Referring to FIG. 1, in order to design a probe molecule that not onlymaintains physiological functions of cholesterol, but also has a largeRaman scattering cross section, we chose to replace the aliphatic sidechain of cholesterol with a cyano or alkynyl groups. These groups havesmall size, which could minimize structural perturbation of the moleculeof interest, in this case, cholesterol. These groups produce strongRaman scattering peaks in a cellular silent region (1,800-2,800 cm⁻¹),and can be used for Raman imaging in a low-concentration condition. Ithas been reported that as the chain length increases, thehyperpolarizability increases in polyynes. Also, aromatic ring cappedalkyne was shown to give stronger Raman signals than terminal alkyne. Todesign cholesterol mimic with very strong Raman intensity, we calculatedthe Raman cross section of potential tags—alkyne, phenyl-alkyne, diyne,phenyl-diyne using the Q-Chem and GAMESS electronic structure packagesto provide insight of the relation between molecular structure and Ramanintensity. The results showed that the localized polarizabilities oneach C≡C moiety increase with the number of conjugated triple bonds, aswell as with addition of a phenyl ring.

The total polarizability of the molecule increases as a result of theadditive effect as well as non-linear boost in the polarizability ofconjugated bonds. The phenyl ring serves as both a donor and an acceptorof π-electrons from the neighboring triple bonds, further escalatingpolarizabilities of neighboring conjugated bonds. Taking into accountthat the Raman intensity is proportional to squares of polarizabilityderivatives, the additional three-fold enhancement of the totalpolarizability due to conjugation results in a ˜10-fold boost in Ramanintensity. Together, the Raman intensity increases further by adding aphenyl group to the terminal alkyne, and increases even further byconjugating a phenyl group and another alkyne.

Raman Spectral Analysis of Stimulate Raman Scattering of CholesterolMimics

Referring now to FIG. 2, to determine the Raman shift of the C≡Cstretching vibrational mode and to compare the level of Raman signalsfrom the cholesterol mimics, 50 mM of each compound was prepared incyclohexanone and confocal Raman spectral analysis was performed. Thesignal from CN-Chol was too weak to be detected. A-Chol showed its peakfor C≡C vibrational mode at 2,122 cm⁻¹; PhA-Chol at 2,239 cm⁻¹;PhDY-Chol at 2,254 cm⁻¹. Comparing the Raman peak of each tag to the1,714 cm⁻¹ C═O vibrational peak from the solvent (9.7 M for purecyclohexanone), the alkyne, PhA, and PhDY groups were 11 times, 16times, and 122 times stronger in Raman intensity, respectively. Thisshowed that the PhDY tag produces a spectrally-isolated peak, which isstronger than the C═O vibrational mode by two orders of magnitude.

To determine the SRS imaging sensitivity for PhDY-Chol, we used afemtosecond stimulated Raman loss (SRL) microscope reported elsewhere.Cyclohexanone solutions of PhDY-Chol were prepared by serial dilution,and SRS images of PhDY-Chol were recorded with the laser beatingfrequency tuned to be resonant with C≡C vibration at 2,252 cm⁻¹. Insolutions without PhDY-Chol, a residual background was detected, causedby cross phase modulation. The SRS contrast, defined as (S−B)/B, where Sand B denote SRS signal and background, was calculated as a function ofPhDY-Chol molar concentration. At the speed of 200 μs per pixel, alinear relationship was observed and 13% and 4% contrasts were reachedat 313 μM and 156 μM, respectively. To increase the detectionsensitivity, we chirped the femtosecond lasers to 0.8 picosecond with aSF-10 glass rod. This spectral focusing approach maintained 85% of theSRS signal while reduced the cross phase modulation background level by3 times, to a level of 6.3×10⁻⁷ in terms of modulation depth. As aresult, the SRS contrast became 14% at 31 μM, corresponding to ˜1,800molecules in the excitation volume. We also depicted the modulationdepth (ΔI/I) as a function of molar concentration (FIG. 8), which isused for estimating the molar concentration of PhDY-Chol inside cells infollowing studies.

Cytoxicity Analysis of the Cholesterol Mimics

Referring now to FIG. 3, the cytotoxicity of cholesterol mimics wasevaluated by MTT cell-viability assays after treating CHO cells withcholesterol mimic. Various concentrations of cholesterol mimic wereadded to the culture media and the cells were incubated for 48 hoursbefore the assays were conducted. A-Chol was found to be toxic to thecells with IC₅₀ of 16 μM. Adding a phenyl group reduced thecytotoxicity. To directly visualize the toxic effect, we stained thecells with propidium iodide for late apoptosis and necrosis. Cellsincubated with A-Chol showed reduced density and extensive apoptosis,whereas both PhA and PhDY caused minimum cell death FIG. 9. This resultpresents another important role of the phenyl group, which is to reducethe toxicity caused by terminal alkyne.

Membrane Incorporation of Cholesterol Mimics

Referring now to FIG. 4, CHO cells which are commonly used forcholesterol trafficking and metabolism studies, were used in thesestudies. To enhance cellular uptake of PhDY-Chol, the cells werepre-incubated in medium supplemented with lipoprotein-deficient serum todeplete medium cholesterol, after which the cells were incubated with 50μM PhDY-Chol for 16 hours. By tuning the laser beating frequency to beresonant with C≡C vibration (2,252 cm⁻¹), SRL signals arose fromPhDY-Chol. We also tuned the laser to be resonant with C—H vibration(2,885 cm⁻¹) and obtained signals from C—H-rich lipid structures, suchas LDs.

To show the incorporation of PhDY-Chol into the plasma membrane, thecells were trypsinized and performed spectral-focusing SRS imaging ofthe rounded live CHO cells with 10 μs per pixel speed. PhDY-Chol in themembrane was detected in the on-resonance image, and the contrastdisappeared in the off-resonance image. The membrane incorporation wasconfirmed by filipin staining of free cholesterol and Raman spectralanalysis (FIG. 10). By focusing at the filipin-stained membrane, we haveobtained the Raman spectrum showing the C═C band from filipin, the amideI band from protein, and the C≡C band from the PhDY (FIG. 10). Insidelive CHO cells, PhDY-Chol was colocalized with LDs found in the C—Hvibrational region, as shown in FIG. 4. This colocalization wasconfirmed by two-photon-excited fluorescence (TPEF) imaging and Ramanspectral analysis of BODIPY-stained LDs in fixed CHO cells. (FIG. 10).The Raman spectrum of the BODIPY-labeled LDs showed the C═C band fromBODIPY, 702 cm⁻¹ peak from cholesterol ring, and the C≡C band from thePhDY (FIG. 11), which further supports the localization of PhDY-Chol inLDs.

It is important to note that PhDY-Chol-rich structures inside the CHOcells could not be stained by filipin (FIG. 12), indicating that it isnot in the free form. It is hypothesized that PhDY-Chol is convertedinto PhDY-cholesteryl ester, by ACAT-1, the enzyme responsible forcholesterol esterification, as diagrammed in FIG. 4. To confirm theesterification of PhDY-Chol, we inhibited ACAT-1 with avasimibe for 24 hbefore addition of PhDY-Chol. After blocking cholesterol esterification,the amount of PhDY-Chol found in CHO cells significantly decreased (FIG.4c ). Although LDs were still visible, the amount of PhDY-Chol signalfound inside LDs reduced by 4 times (FIG. 4d ). ACAT-1 knockdown byshRNA was also conducted to specifically inhibit the enzyme. Similarly,we found decreased amount of PhDY-Chol in ACAT-1 knocked down CHO cells,and the amount of PhDY-Chol in LDs reduced significantly (FIG. 12). Todetermine where PhDY-Chol accumulates after ACAT-1 inhibition, westained the cells with LysoTracker for lysosomes. Our results indicatedthat after ACAT-1 inhibition, PhDY-Chol partially located in lysosomes(FIG. 12). Collectively, these results show that PhDY-Chol can betransported into cells, converted into PhDY-cholesteryl ester by ACAT-1,and stored in LDs following the normal metabolic pathway of cholesterol.To emphasize the physiological compatibility of our PhDY tag, we treatedCHO cells with BODIPY-cholesterol. The amount of BODIPY-cholesterolincorporated into LDs did not change after ACAT-1 inhibition (FIGS. 4dand e ), indicating that BODIPY-cholesterol directly labels the LDswithout metabolic conversion into cholesteryl ester.

Lysosomal accumulation and relocation to Lipid Droplets in NP-C animaldisease model discovery using cholesterol mimics.

Referring now to FIG. 5, the potential of PhDY-Chol for studyingcholesterol transport in NP-C disease, a disorder featured by abnormalcholesterol accumulation in late endosome/lysosome caused by mutation inNPC1 or 2 gene, was explored. M12 cells, mutant CHO cells that contain adeletion of the NPC1 locus, were established as a cellular model of theNP-C disease. By combining SRL imaging of PhDY with TPEF imaging offilipin, we observed that, unlike wildtype CHO cells, the PhDY-Chol-richstructures were stained by filipin, indicating that these PhDY-Cholmolecules were located in lysosomes. (FIG. 13). Moreover, we observedsome filipin labeled structures that do not contain PhDY-Chol. Thisresult is reasonable given that filipin has been shown to label otherlipid molecules, such as glycosphingolipids. As additional evidence, weincubated M12 cells with PhDY-Chol and stained the cells withLysoTracker. It was found that all PhDY-Chol-rich areas were localizedin LysoTracker-stained organelles (FIG. 13). Collectively, these resultsshowed that PhDY-Chol can selectively represent the lysosomal storage ofcholesterol in the NP-C disease model.

We then treated the PhDY-Chol-labeled M12 cells with acholesterol-mobilizing drug, hydroxypropyl-β-cyclodextrin (HPβCD). Thisdrug is known to mediate lysosomal escape of cholesterol, and promotestorage of excess cholesterol into LDs. After treating with HPβCD, theamount of PhDY-Chol in M12 cells decreased by half (FIGS. 5b and c ).Interestingly, we observed that some PhDY-Chol-rich areas were notlabeled by filipin after HPβCD treatment (FIG. 5b , arrow heads). Theseareas likely represent PhDY-cholesteryl ester stored in LDs. To confirmthis possibility, we stained the cells with BODIPY for localization ofLDs. The result clearly showed that PhDY-Chol moved into LDs after HPβCDtreatment, and the number of PhDY-rich LDs increased significantly(FIGS. 5d and e ). Together, these data indicate that PhDY-Chol can beused as a reliable probe molecule to study cholesterol mobilizationinside living cells.

Cholesterol mimics identify cholesterol storage compartments in animalmodel.

Referring to FIG. 6, to demonstrate the capability of monitoringcholesterol uptake and distribution in vivo, we fed N2 wildtype C.elegans with PhDY-Chol-labeled E. coli and imaged PhDY-Chol storage inthe worms using our SRL microscope at speed of 40 μs per pixel.PhDY-Chol was found to be stored in the intestinal cells inside thewildtype worms (FIG. 6a , upper panels). To confirm the uptake ofPhDY-Chol by intestinal cells, we fed mutant C. elegans, in whichdietary cholesterol uptake is inhibited by ChUP-1 deletion, withPhDY-Chol. We did not observe PhDY-Chol inside this strain (FIG. 6a ,lower panels), which indicates that the PhDY tag did not affect thecholesterol uptake process. Then, we tuned the laser to be resonant withC—H vibration for lipid-rich LDs. Unlike wildtype CHO cells, thePhDY-Chol-rich compartments were found to be distinguished from LDs inwildtype worms (FIG. 6a , upper panels). To explore the nature of thesecompartments, we used hjIs9 worms that contains GFP targeted tolysosome-related organelles (LROs) in intestinal cells. Dual-modalitySRS and TPEF imaging showed that PhDY-Chol is stored in the LROs (FIG.6b ). Collectively, these results suggest that dietary PhDY-Chol uptakeis through a ChUP-1 mediated process, and unlike mammalian CHO cells, C.elegans stores cholesterol in LROs, but not in LDs in the intestine.

Chemical structure (V) or Compound S8. The mixture of 4 (22 mg, 0.05mmol), PdCl₂(PPh₃)₂(1.4 mg, 0.002 mmol), CuI (0.4 mg, 0.002 mmol), and B(12.3 mg, 0.06 mmol) in THF (0.5 mL) was bubbled with Argon gas for tenminutes. To the mixture, DIPEA (0.02 mL) was added at room temperature.After stirring for 2 h at room temperature, the reaction was quenchedwith saturated NH₄Cl aqueous solution and extracted with ethyl acetate.The organic layer was washed with brine, dried over MgSO₄. The solventwas removed under vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 40:1) to give S8 (18 mg, 65%) as a white solid.

¹H NMR (500 MHz, CDCl₃): δ 7.50 (d, J=7.0 Hz, 2H), 7.38-7.35 (m, 1H),7.33-7.30 (m, 2H), 5.35 (t, J=6.5 Hz, 1H), 4.72 (m, 1H), 3.93-3.90 (m,1H), 3.55-3.47 (m, 2H), 2.44-2.20 (m, 4H), 2.01-1.96 (m, 2H), 1.88-1.83(m, 4H), 1.74-1.70 (m, 2H), 1.62-1.42 (m, 12H), 1.30-1.06 (m, 7H), 1.01(s, 3H), 0.93 (d, J=6.5 Hz, 3H), 0.69 (s, 3H); ¹³C NMR (100 MHz, CDCl₃):δ 141.2, 141.1, 133.1, 129.6, 128.6, 121.7, 121.6, 121.4, 108.8, 97.2,97.0, 83.3, 76.2, 75.5, 74.8, 67.6, 65.6, 63.1, 63.0, 59.6, 56.9, 56.0,53.6, 50.3, 50.3, 42.6, 40.4, 40.0, 38.9, 37.6, 37.4, 37.0, 36.9, 35.4,34.5, 32.0, 31.4, 29.8, 28.3, 28.1, 25.6, 24.4, 21.2, 20.3, 20.2, 19.5,18.3, 16.8, 12.0; IR (film): 2958, 2925, 2326, 2125, 1643, 1457, 1379,1016 cm⁻¹; MS (ESI): m/z 585 [M+Na]⁺.

Additional disclosure and drawings can be found in the followingparagraphs and updated drawings submitted herewith. They are part of theprovisional application with the Ser. No. 62/048,484 that the instantapplication claims benefits from and incorporated herein entirely.

Rational Design and Synthesis of Tagged Cholesterol with an ExtremelyLarge Raman Scattering Cross Section

In order to design a probe molecule that not only maintainsphysiological functions of cholesterol, but also has a large Ramanscattering cross section, we chose to replace the aliphatic side chainof cholesterol with a cyano or alkynyl groups (FIG. 1). These groupshave small size, which could minimize structural perturbation of themolecule of interest, in this case, cholesterol. Meanwhile, these groupsproduce strong Raman scattering peaks in a cellular silent region(1,800-2,800 cm⁻¹), and therefore, can potentially be used for Ramanimaging in a low-concentration condition. It has been reported that asthe chain length increases, the hyperpolarizability increases inpolyynes. Also, aromatic ring capped alkyne was shown to give strongerRaman signals than terminal alkyne. To design tagged cholesterol withvery strong Raman intensity, we calculated the Raman cross section ofpotential tags—alkyne, phenyl-alkyne, diyne, phenyl-diyne using theQ-Chem and GAMESS electronic structure packages to provide insight ofthe relation between molecular structure and Raman intensity. Ourresults show that the localized polarizabilities on each C≡C moietyincrease with the number of conjugated triple bonds, as well as withaddition of a phenyl ring (FIG. 7a and Table 1). Thus, the totalpolarizability of the molecule increases as a result of the additiveeffect as well as non-linear boost in the polarizability of conjugatedbonds. The phenyl ring serves as both a donor and an acceptor ofπ-electrons from the neighboring triple bonds, further escalatingpolarizabilities of neighboring conjugated bonds. Taking into accountthat the Raman intensity is proportional to squares of polarizabilityderivatives, the additional three-fold enhancement of the totalpolarizability due to conjugation results in a ˜10-fold boost in Ramanintensity. Together, the Raman intensity increases by 9 times by addinga phenyl group to the terminal alkyne, and 52 times by conjugating aphenyl group and another alkyne (FIG. 7b ).

Based on the above considerations, we have synthesized a series oftagged cholesterols—alkyne cholesterol (A-Chol, 5), phenyl-alkynecholesterol (PhA-Chol, 6), phenyl-diyne cholesterol (PhDY-Chol, 7), andcyano cholesterol (CN-Chol, 8) as shown in FIG. 1. Our synthesiscommenced with commercially available cholenic acid 3. Using a sequenceof THP-protection, LiAlH₄ reduction, Dess-Martin oxidation andSeyferth-Gilbert-Bestmann homologation, cholenic acid 3 was converted tocompound 4 with a terminal alkyne group in excellent yield. Removal ofthe THP-protecting group gave probe 5. We further prepared PhA-Chol 6and PhDY-Chol 7 from compound 4 via a palladium-catalyzed Sonogashirareaction and a copper-catalyzed Cadiot-Chodkiewicz reaction,respectively, followed by acidic removal of THP group. Additionally,CN-Chol 8 was prepared from cholenic acid 3 via standardtransformations.

Raman Spectral Analysis and SRS Imaging of Tagged Cholesterol

To determine the Raman shift of the C≡C stretching vibrational mode andto compare the level of Raman signals from the tagged cholesterols, weprepared 50 mM of each compound in cyclohexanone and performed confocalRaman spectral analysis (FIG. 2a ). The signal from CN-Chol was too weakto be detected. A-Chol showed its peak for C≡C vibrational mode at 2,122cm⁻¹; PhA-Chol at 2,239 cm⁻¹; PhDY-Chol at 2,254 cm⁻¹. (FIG. 2a ).Comparing the Raman peak of each tag to the 1,714 cm⁻¹ C═O vibrationalpeak from the solvent (9.7 M for pure cyclohexanone), the alkyne, PhA,and PhDY groups were 11 times, 16 times, and 122 times stronger in Ramanintensity, respectively. (FIG. 2b ). This result showed that the PhDYtag produces a spectrally-isolated peak, which is stronger than the C═Ovibrational mode by two orders of magnitude.

To determine the SRS imaging sensitivity for PhDY-Chol, we used afemtosecond stimulated Raman loss (SRL) microscope reported elsewhere²⁸.Cyclohexanone solutions of PhDY-Chol were prepared by serial dilution,and SRS images of PhDY-Chol were recorded with the laser beatingfrequency tuned to be resonant with C≡C vibration at 2,252 cm⁻¹. Insolutions without PhDY-Chol, a residual background was detected, causedby cross phase modulation. The SRS contrast, defined as (S−B)/B, where Sand B denote SRS signal and background, was calculated as a function ofPhDY-Chol molar concentration. At the speed of 200 μs per pixel, alinear relationship was observed (FIG. 2c ) and 13% and 4% contrastswere reached at 313 μM and 156 μM, respectively. To increase thedetection sensitivity, we chirped the femtosecond lasers to 0.8picosecond with a SF-10 glass rod. This spectral focusing approachmaintained 85% of the SRS signal while reduced the cross phasemodulation background level by 3 times, to a level of 6.3×10⁻⁷ in termsof modulation depth. As a result, the SRS contrast became 14% at 31 μM,corresponding to ˜1,800 molecules in the excitation volume (FIG. 2d ).We also depicted the modulation depth (ΔI/I) as a function of molarconcentration (FIG. 8), which is used for estimating the molarconcentration of PhDY-Chol inside cells in following studies.

Cytotoxicity Caused by Terminal Alkyne is Avoided by Phenyl Group

To evaluate the cytotoxicity of tagged cholesterols, we performed MTTcell-viability assays after treating CHO cells with tagged cholesterol.Various concentrations of tagged cholesterol were added to the culturemedia and the cells were incubated for 48 h before the assays wereconducted. A-Chol was found to be toxic to the cells with IC₅₀ of 16 μM.Importantly, adding a phenyl group effectively reduced the cytotoxicity(FIG. 3). To directly visualize the toxic effect, we stained the cellswith propidium iodide for late apoptosis and necrosis. Cells incubatedwith A-Chol showed reduced density and extensive apoptosis, whereas bothPhA and PhDY caused minimum cell death (FIG. 9). This result presentsanother important role of the phenyl group, which is to reduce thetoxicity caused by terminal alkyne. Based on the signal level and theseverity of toxicity, we conclude that PhDY-Chol is the most suitablecholesterol probe for live cell imaging, and we used PhDY-Chol insubsequent experiments.

Membrane Incorporation and Esterification of PhDY-Chol in Live Cells

We chose CHO cells which are commonly used for cholesterol traffickingand metabolism studies. To enhance cellular uptake of PhDY-Chol, thecells were pre-incubated in medium supplemented withlipoprotein-deficient serum to deplete medium cholesterol, after whichthe cells were incubated with 50 μM PhDY-Chol for 16 h. By tuning thelaser beating frequency to be resonant with C≡C vibration (2,252 cm⁻¹),SRL signals arose from PhDY-Chol. We also tuned the laser to be resonantwith C—H vibration (2,885 cm⁻¹) and obtained signals from C—H-rich lipidstructures, such as LDs.

To show the incorporation of PhDY-Chol into the plasma membrane, wetrypsinized the cells and performed spectral-focusing SRS imaging of therounded live CHO cells with 10 μs per pixel speed. PhDY-Chol in themembrane was detected in the on-resonance image, and the contrastdisappeared in the off-resonance image (FIG. 10a ). The membraneincorporation was confirmed by filipin staining of free cholesterol andRaman spectral analysis (FIGS. 10b and c ). By focusing at thefilipin-stained membrane, we have obtained the Raman spectrum showingthe C═C band from filipin, the amide I band from protein, and the C≡Cband from the PhDY (FIG. 10c ). Inside live CHO cells, PhDY-Chol wascolocalized with LDs found in the C—H vibrational region (FIG. 4a ).This colocalization was confirmed by two-photon-excited fluorescence(TPEF) imaging and Raman spectral analysis of BODIPY-stained LDs infixed CHO cells. (FIGS. 11a and b ). The Raman spectrum of theBODIPY-labeled LDs showed the C═C band from BODIPY, 702 cm⁻¹ peak fromcholesterol ring, and the C≡C band from the PhDY (FIG. 11b ), whichfurther supports the localization of PhDY-Chol in LDs.

It is important to note that PhDY-Chol-rich structures inside the CHOcells could not be stained by filipin (FIG. 10b ), implicating that itis not in the free form. We hypothesize that PhDY-Chol is converted intoPhDY-cholesteryl ester, by ACAT-1, the enzyme responsible forcholesterol esterification (FIG. 4b ). To confirm the esterification ofPhDY-Chol, we inhibited ACAT-1 with avasimibe for 24 h before additionof PhDY-Chol. After blocking cholesterol esterification, the amount ofPhDY-Chol found in CHO cells significantly decreased (FIG. 4c ).Although LDs were still visible, the amount of PhDY-Chol signal foundinside LDs reduced by 4 times (FIG. 4d ). ACAT-1 knockdown by shRNA wasalso conducted to specifically inhibit the enzyme. Similarly, we founddecreased amount of PhDY-Chol in ACAT-1 knocked down CHO cells, and theamount of PhDY-Chol in LDs reduced significantly (FIG. 12a ). Todetermine where PhDY-Chol accumulates after ACAT-1 inhibition, westained the cells with LysoTracker for lysosomes. Our result indicatedthat after ACAT-1 inhibition, PhDY-Chol partially located in lysosomes(FIG. 12b ). Collectively, these results show that PhDY-Chol can betransported into cells, converted into PhDY-cholesteryl ester by ACAT-1,and stored in LDs following the normal metabolic pathway of cholesterol.To emphasize the physiological compatibility of our PhDY tag, we treatedCHO cells with BODIPY-cholesterol. The amount of BODIPY-cholesterolincorporated into LDs did not change after ACAT-1 inhibition (FIGS. 4dand 4e ), indicating that BODIPY-cholesterol directly labels the LDswithout metabolic conversion into cholesteryl ester.

Lysosomal Accumulation and Relocation to LDs in NP-C Disease Model

Next, we explored the potential of PhDY-Chol for studying cholesteroltransport in NP-C disease, a disorder featured by abnormal cholesterolaccumulation in late endosome/lysosome caused by mutation in NPC1 or 2gene³⁸. M12 cells, mutant CHO cells that contain a deletion of the NPC1locus, were established as a cellular model of the NP-C disease³⁹. Bycombining SRL imaging of PhDY with TPEF imaging of filipin, we observedthat, unlike wildtype CHO cells, the PhDY-Chol-rich structures werestained by filipin, indicating that these PhDY-Chol molecules werelocated in lysosomes. (FIG. 5a , FIGS. 13a and 13b ). Moreover, weobserved some filipin labeled structures that do not contain PhDY-Chol.This result is reasonable given that filipin has been shown to labelother lipid molecules, such as glycosphingolipids²¹. As additionalevidence, we incubated M12 cells with PhDY-Chol and stained the cellswith LysoTracker. It was found that all PhDY-Chol-rich area werelocalized in LysoTracker-stained organelles (FIG. 13c ). Collectively,these results showed that PhDY-Chol can selectively represent thelysosomal storage of cholesterol in the NP-C disease model.

We then treated the PhDY-Chol-labeled M12 cells with acholesterol-mobilizing drug, HPβCD⁴⁰. This drug is known to mediatelysosomal escape of cholesterol, and promote storage of excesscholesterol into LDs⁴¹. After treating with HPβCD, the amount ofPhDY-Chol in M12 cells decreased by half (FIGS. 5b and c ).Interestingly, we observed that some PhDY-Chol-rich area were notlabeled by filipin after HPβCD treatment (FIG. 5b , arrow heads). Theseareas likely represent PhDY-cholesteryl ester stored in LDs. To confirmthis possibility, we stained the cells with BODIPY for localization ofLDs. The result clearly showed that PhDY-Chol has moved into LDs afterHPβCD treatment, and the number of PhDY-rich LDs increased significantly(FIGS. 5d and e ). Together, these data indicate that PhDY-Chol can beused as a reliable probe molecule to study cholesterol mobilizationinside living cells.

Cholesterol Storage Compartments in C. elegans Visualized by PhDY-Chol

Finally, to demonstrate the capability of monitoring cholesterol uptakeand distribution in vivo, we fed N2 wildtype C. elegans withPhDY-Chol-labeled E. coli and imaged PhDY-Chol storage in the wormsusing our SRL microscope at speed of 40 s per pixel. PhDY-Chol was foundto be stored in the intestinal cells inside the wildtype worms (FIG. 6a, upper panels). To confirm the uptake of PhDY-Chol by intestinal cells,we fed mutant C. elegans, in which dietary cholesterol uptake isinhibited by ChUP-1 deletion, with PhDY-Chol. We did not observePhDY-Chol inside this strain (FIG. 6a , lower panels), which indicatesthat the PhDY tag did not affect the cholesterol uptake process. Then,we tuned the laser to be resonant with C—H vibration for lipid-rich LDs.Unlike wildtype CHO cells, the PhDY-Chol-rich compartments were found tobe distinguished from LDs in wildtype worms (FIG. 6a , upper panels). Toexplore the nature of these compartments, we used hjIs9 worms thatcontains GFP targeted to lysosome-related organelles (LROs) inintestinal cells⁴³. Dual-modality SRS and TPEF imaging showed thatPhDY-Chol is stored in the LROs (FIG. 6b ). Collectively, these resultssuggest that dietary PhDY-Chol uptake is through a ChUP-1 mediatedprocess, and unlike mammalian CHO cells, C. elegans stores cholesterolin LROs, but not in LDs in the intestine. We have developed a series oftagged cholesterols based on quantum chemistry calculations and chemicalsynthesis. By using PhDY to replace the aliphatic chain in cholesterol,we produced a cholesterol probe, PhDY-Chol, with a Raman signal that istwo orders of magnitude stronger than the C═O group. By SRS imaging oflive CHO cells, PhDY-Chol was found to be incorporated into themembrane, and converted to PhDY-cholesteryl ester for storage in LDs.With this cholesterol probe, we experimentally validated that afterACAT-1 inhibition, cholesterol partly accumulates in lysosomes. In liveNPC1-deleted CHO cells, PhDY-Chol selectively represented lysosomalaccumulation of cholesterol in untreated cells, and esterification andrelocation to LDs after HPβCD treatment. Lastly, SRS imaging ofPhDY-Chol in live C. elegans identified LROs, but not LDs, as thecholesterol storage compartments in the intestine.

In this study, we have developed a series of tagged cholesterols basedon quantum chemistry calculations and chemical synthesis. By using PhDYto replace the aliphatic chain in cholesterol, we produced a cholesterolprobe, PhDY-Chol, with a Raman signal that is two orders of magnitudestronger than the C═O group. By SRS imaging of live CHO cells, PhDY-Cholwas found to be incorporated into the membrane, and converted toPhDY-cholesteryl ester for storage in LDs. With this cholesterol probe,we experimentally validated that after ACAT-1 inhibition, cholesterolpartly accumulates in lysosomes. In live NPC1-deleted CHO cells,PhDY-Chol selectively represented lysosomal accumulation of cholesterolin untreated cells, and esterification and relocation to LDs after HPβCDtreatment. Lastly, SRS imaging of PhDY-Chol in live C. elegansidentified LROs, but not LDs, as the cholesterol storage compartments inthe intestine.

Essential parameters of a valid Raman tag include its amplitude of Ramanscattering cross section, cytotoxicity, and biocompatibility. Althoughthe C-D bond can be used to replace C—H bonds without changing thestructures of the molecules, it gives relatively weak Raman intensities.Raman signal from alkyne bond is stronger than that from C-D bond by oneorder of magnitude, and detection at hundreds of μM of alkyne-containingmolecules by SRS microscopy was reported. In our study, through rationaldesign and synthesis of a PhDY tag, we increased the Raman scatteringcross section by 11 times compared to the alkyne group, and 122 timescompared to the endogenous C═O group. This enhancement is a result ofconjugation of π-electrons among the two C≡C bonds and the phenyl group.As a result, we have been able to detect ˜30 μM of PhDY-Chol molecules(˜1,800 molecules at excitation volume), and demonstrated SRS imaging ofPhDY-Chol in single membrane at speed of 10 μs per pixel. Importantly,this design also shielded the activity of terminal alkyne andsignificantly reduced cytotoxicity. Moreover, PhDY-Chol structurallymimics cholesterol, using the same physiological process for cholesteroltransport and metabolism inside cells. Using the same strategy, otherRaman tag molecules can be designed for sensitive and biocompatibleprobing of biomolecules in live cells.

The potential value of a Raman tag is also related to the detectionsensitivity of SRS microscopy. One limitation comes from the cross phasemodulation, which produces a background that reduces the contrast forthe tag molecules. Although broadband femtosecond lasers provide highpeak intensity to enhance the SRS signal, they also increase theamplitude of the cross phase modulation. As shown in our study, thisbackground can be reduced by 3 times using spectral focusing. Thespectral focusing approach also increases the spectral selectivity,reduces the photodamage, and provides opportunities to conducthyperspectral SRS imaging.

In this study, we compared BODIPY-cholesterol and PhDY-Chol. Our resultsshow that PhDY-Chol is stored in LDs via esterification which can beblocked by ACAT-1 inhibition. In contrast, BODIPY-cholesterol labels LDseven after ACAT-1 inhibition. This result may be due to the stronghydrophobic interaction of BODIPY with LDs, and is consistent withprevious studies showing that BODIPY-cholesterol is hardly esterified byACAT-1 inside the cells. We also showed that PhDY-Chol reflects thelocation of the cholesterol more specifically than filipin staining.Lastly, SRS microscopy utilizes chemical-bond vibrational signals forvisualization. Thus, unlike fluorophores, the PhDY tag does not undergobleaching (FIG. 14), in contrast to BODIPY-cholesterol and DHE which isknown to have a rapid photo-bleaching rate. Combining these uniqueproperties, PhDY-Chol allows quantitative imaging of intracellularcholesterol, and repetitive observation of the same sample before andafter treatment.

It is worth discussing the new opportunities for study of the NP-Cdisease and genomic screening of cholesterol-related genes. NP-C diseaseis a fatal neurodegenerative disease that show extensive lysosomalaccumulation of cholesterol, and early detection and treatmentstrategies are still under development. The involvement of lysosomalcholesterol accumulation to the neurodegeneration is still unclear. Inthis study, we have demonstrated in vitro study of cholesteroltrafficking and metabolism in a cellular model. This can be extended toin vivo studies using mouse models to understand the progression of thedisease and impact of potential therapeutic strategies, especially incentral nervous system. C. elegans is an important model for genetic andchemical screening in many diseases, including NP-C disease. It has beenproposed that the intracellular sterol trafficking pathway might beconserved in this animal model. However, imaging cholesterol in C.elegans has been a challenge due to strong autofluorescence from theworm. SRS imaging of PhDY-Chol opens an avenue to genome-wide RNAinterference screening of C. elegans for cholesterol transport andstorage genes in this animal model. Finally, our work also opens newopportunities to study cholesterol trafficking and metabolism in otheranimal models such as zebrafish and mice.

Methods

Calculation of Raman Intensity.

All calculations were performed at the HF/6-311G* level of theory.Geometry optimizations, vibrational frequencies and Raman intensitiesare obtained in Q-Chem electronic structure package. Localizedpolarizabilities are calculated in the GAMESS quantum chemistrysoftware.

Chemicals.

3β-hydroxy-Δ⁵-cholenic acid was purchased from VWR.Lipoprotein-deficient serum was purchased from Biomedical TechnologiesInc. Cholesterol, avasimibe, and filipin complex were purchased fromSigma-Aldrich. BODIPY-cholesterol was purchased from Avanti PolarLipids, Inc. Propidium iodide, BODIPY, and LysoTracker were purchasedfrom Life technologies.

Synthesis of Raman-Tagged Cholesterol.

Detailed synthesis procedures and characterization of compounds areshown as following:

Synthesis of Cholesterol Mimics

General Methods. NMR spectra were recorded on (¹H at 400 MHz, 500 MHzand ¹³C at 100 MHz, 125 MHz) spectrometers, Chemical shifts (5) weregiven in ppm with reference to solvent signals [¹H NMR: CDCl₃ (7.26);¹³C NMR: CDCl₃ (77.2)]. Column chromatography was performed on silicagel. All reactions sensitive to air or moisture were carried out underargon atmosphere in dry and freshly distilled solvents under anhydrousconditions, unless otherwise noted. Anhydrous THF was distilled oversodium benzophenone ketyl under N₂. Anhydrous CH₂Cl₂ was distilled overcalcium hydride under N₂. Anhydrous MeOH was distilled over magnesiumunder N₂. All other solvents and reagents were used as obtained fromcommercial sources without further purification.

Compound S1.

To a solution of acid 3 (200 mg, 0.53 mmol) in dry THF (22.5 mL) wereadded 3,4-dihydro-2H-pyran (DHP) (0.24 mL, 2.65 mmol) andp-toluenesulfonic acid monohydrate (p-TsOH) (20.2 mg, 0.11 mmol) underargon. After the mixture was stirred at room temperature for 24 h,saturated aqueous NaHCO₃ solution and CH₂Cl₂ were added to quench thereaction. The aqueous layer was extracted with CH₂Cl₂ (3×50 mL), and thecombined organic layers were acidified with acetic acid (15 mL), washedwith water (3×50 mL) and dried with Na₂SO₄. The solvent was removedunder vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 8:1) to give S1 (222 mg, 91%) as a white solid.

¹H NMR (500 MHz, CDCl₃): δ 5.35-5.33 (m, 1H), 4.74-4.71 (m, 1H),3.93-3.90 (m, 1H), 3.54-3.46 (m, 1H), 2.42-2.19 (m, 4H), 1.98-1.71 (m,9H), 1.61-1.43 (m, 12H), 1.35-1.07 (m, 7H), 1.00 (s, 3H), 0.93 (d, J=7.0Hz, 3H), 0.67 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 180.0, 141.2, 121.7,97.1, 96.9, 76.2, 63.0, 62.9, 56.9, 55.9, 50.3, 42.5, 40.4, 39.9, 38.9,37.6, 37.4, 36.9, 35.5, 32.0, 31.4, 31.1, 30.9, 29.8, 28.2, 28.2, 28.1,25.6, 24.4, 21.2, 20.1, 19.5, 18.4, 12.0; IR (film): 2938, 1708, 1454,1200, 1059, 1033, 975 cm⁻¹; MS (ESI): m/z 457.4 [M-H]⁻.

Compound S2.

A solution of S1 (222 mg, 0.48 mmol) in dry THF (12 mL) was addeddropwise to a suspension of LiAlH₄ (55 mg, 1.45 mmol) in dry THF (8 mL)under argon at 0° C. After the addition, the reaction mixture wasallowed to warm to room temperature and stirred overnight. Saturatedaqueous NaHCO₃ solution (10 mL) was added slowly to quench the reaction,and the resulting mixture was extracted with CH₂Cl₂ (3×60 mL). Thecombined organic layers were washed with brine and dried with Na₂SO₄.The solvent was removed under vacuum, and the residue was purified bychromatography (Hexane/EtOAc, 4:1) to give S2 (209 mg, 98%) as a whitesolid.

¹H NMR (400 MHz, CDCl₃): δ 5.32 (t, J=6.0 Hz, 1H), 4.70 (s, 1H),3.93-3.86 (m, 1H), 3.58 (td, J=9.2, 2.8 Hz, 2H), 3.52-3.45 (m, 2H),2.34-2.18 (m, 2H), 1.97-1.81 (m, 8H), 1.57-1.38 (m, 16H), 1.23-1.05 (m,6H), 0.98 (s, 3H), 0.92 (d, J=6.4 Hz, 3H), 0.66 (s, 3H); ¹³C NMR (100MHz, CDCl₃): δ 141.2, 141.0, 121.6, 121.6, 97.1, 96.9, 76.1, 63.6, 63.0,62.9, 56.8, 56.1, 50.3, 50.2, 42.4, 40.3, 39.9, 38.9, 37.6, 37.3, 36.9,36.9, 35.7, 32.0, 32.0, 31.4, 29.8, 29.5, 28.3, 28.1, 25.6, 24.4, 21.2,20.2, 20.1, 19.5, 18.8, 12.0; IR (film): 2936, 1456, 1377, 1112, 1059,1033 cm⁻¹; MS (ESI): m/z 467.4 [M+Na]⁺.

Compound S3.

To a suspension of S2 (79 mg, 0.18 mmol) and NaHCO₃ (45 mg, 0.53 mmol)in CH₂Cl₂ (4 mL) was added freshly prepared Dess-Martin Periodinane (113mg, 0.27 mmol) at 0° C. The reaction mixture was stirred at 0° C. Afterthe reaction is complete, saturated aqueous NaHCO₃ solution (10 mL) andsaturated aqueous Na₂S₂O₃ solution (10 mL) were added and allowed tostir for 30 min at room temperature. Two layers were separated and theaqueous layer was washed with EtOAc (2×40 mL). The combined organiclayers were washed with saturated aqueous NaHCO₃ solution (2×30 mL),brine and dried with Na₂SO₄. The solvent was removed under vacuum, andthe residue was purified by chromatography (Hexane/EtOAc, 4:1) to giveS3 (68 mg, 86%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 9.75 (t, J=2.0 Hz, 1H), 5.33 (td, J=5.2, 1.6Hz, 1H), 4.70 (t, J=3.6 Hz, 1H), 3.93-3.87 (m, 1H), 3.55-3.44 (m, 2H),2.44-2.18 (m, 4H), 1.99-1.93 (m, 2H), 1.84-1.69 (m, 6H), 1.57-1.42 (m,12H), 1.33-1.05 (m, 7H), 0.99 (s, 3H), 0.91 (d, J=6.4 Hz, 3H), 0.66 (s,3H); ¹³C NMR (100 MHz, CDCl₃): δ 203.2, 141.2, 142.0, 121.6, 121.5,97.1, 96.9, 76.1, 63.0, 63.0, 56.8, 55.9, 50.2, 50.2, 42.5, 41.0, 40.4,39.8, 38.9, 37.6, 37.3, 36.9, 36.9, 35.4, 32.0, 31.4, 29.8, 28.3, 28.1,25.6, 24.4, 21.1, 20.2, 20.1, 19.5, 18.5, 12.9; IR (film): 2936, 2867,1726, 1440, 1199, 1134, 1033, 1025, 976 cm⁻¹; MS (ESI): m/z 441.1[M-H]⁻.

Compound 4.

To a solution of S3 (415 mg, 0.914 mmol) and K₂CO₃ (504.6 mg, 3.66 mmol)in dry methanol (14 mL) and THF (14 mL) were addeddimethyl-1-diazo-2-oxopropylphosphonate (Bestmann Reagent, 0.33 mL,2.194 mmol) and stirred under room temperature. After the reaction wascomplete, the reaction was diluted with EtOAc, washed with saturatedaqueous NaHCO₃ solution, and dried over MgSO₄. The solvent was removedunder vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 20:1) to give 4 (397 mg, 99%) as a white solid.

¹H NMR (500 MHz, CDCl₃): δ 5.35-5.32 (m, 1H), 4.71-4.70 (m, 1H),3.93-3.88 (m, 1H), 3.53-3.46 (m, 2H), 2.35-2.09 (m, 4H), 2.00-1.82 (m,7H), 1.73-1.70 (m, 2H), 1.64-1.40 (m, 12H), 1.30-1.05 (m, 7H), 1.00 (s,3H), 0.92 (d, J=6.5 Hz, 3H), 0.68 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ141.2, 141.0, 121.6, 121.6, 97.1, 96.9, 85.3, 76.1, 68.0, 63.0, 62.9,56.8, 56.0, 50.3, 50.2, 42.6, 40.4, 39.9, 38.9, 37.6, 37.3, 36.9, 36.9,35.3, 35.0, 32.0, 32.0, 31.4, 28.3, 28.1, 25.6, 24.4, 21.2, 20.2, 20.2,19.5, 18.3, 15.6, 12.0; IR (film): 3311, 2935, 2868, 2850, 2118, 1466,1454, 1440, 1376, 1114, 1057, 869 cm⁻¹; MS (ESI): m/z 461.4 [M+Na]⁺.

Compound S4.

To a solution of S2 (100 mg, 0.225 mmol) and triethylamine (0.094 mL,0.676 mmol) in CH₂Cl₂ (5 mL) was added methanesulfonyl chloride (77.4mg, 0.676 mmol) under 0° C. After the addition, the reaction mixture wasallowed to warm to room temperature and stirred overnight. Saturatedaqueous NaHCO₃ solution was added to quench the reaction, and theresulting mixture was extracted with ethyl ether. The combined organiclayers were washed with brine and dried with MgSO₄. The solvent wasremoved under vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 8:1) to give 7 (88 mg, 75%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 5.33 (t, J=4.8 Hz, 1H), 4.70 (s, 1H), 4.19(dd, J=10.0, 5.2 Hz, 2H), 3.92-3.89 (m, 1H), 3.54-3.46 (m, 2H), 2.99 (s,3H), 2.35-2.16 (m, 2H), 2.00-1.94 (m, 2H), 1.85-1.79 (m, 5H), 1.70-1.44(m, 15H), 1.24-1.08 (m, 7H), 1.00 (s, 3H), 0.93 (d, J=5.2 Hz, 3H), 0.67(s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 141.2, 141.0, 121.6, 121.6, 97.1,97.0, 76.1, 70.8, 63.0, 63.0, 56.8, 55.9, 50.2, 42.5, 40.4, 39.9, 38.9,37.5, 37.3, 36.9, 35.4, 32.0, 31.6, 31.4, 29.8, 28.3, 28.1, 26.0, 25.6,25.6, 24.4, 21.2, 20.2, 19.5, 18.7, 12.0; IR (film): 2937, 2881, 2858,1355, 1034, 962, 837 cm⁻¹; MS (ESI): m/z 545.4 [M+Na]⁺.

Compound S5.

To a solution of S4 (40 mg, 0.077 mmol) in DMSO (1.5 mL) was addedpotassium cyanide (10 mg, 0.153 mmol). After the addition, the reactionmixture was heated to 90° C. and stirred for 2 h. Water (5 mL) was addedto quench the reaction, and the resulting mixture was extracted withethyl acetate (4×4 mL). The combined organic layers were washed withbrine and dried with MgSO₄. The solvent was removed under vacuum, andthe residue was purified by chromatography (Hexane/EtOAc, 8:1) to giveS5 (27 mg, 76%) as a white solid.

¹H NMR (500 MHz, CDCl₃): δ 5.34 (dd, J=6.8, 4.4 Hz, 1H), 4.72-4.70 (m,1H), 3.94-3.89 (m, 1H), 3.55-3.46 (m, 2H), 2.36-2.16 (m, 4H), 2.00-1.94(m, 2H), 1.87-1.83 (m, 4H), 1.73-1.68 (m, 2H), 1.62-1.41 (m, 14H),1.30-1.04 (m, 7H), 1.00 (s, 3H), 0.93 (d, J=6.5 Hz, 3H), 0.68 (s, 3H);¹³C NMR (125 MHz, CDCl₃): δ 141.2, 141.0, 121.6, 121.6, 120.0, 97.1,97.0, 76.1, 63.1, 63.0, 56.8, 56.0, 50.3, 50.2, 42.5, 40.4, 39.9, 39.0,37.6, 37.3, 36.9, 36.9, 35.4, 35.2, 32.0, 31.4, 29.8, 28.3, 28.1, 25.6,24.4, 22.3, 21.2, 20.2, 20.2, 19.5, 18.7, 17.7, 12.0; IR (film): 2924,2854, 2352, 2323, 1456, 1033, 1021, 973 cm⁻¹; MS (ESI): m/z 476.4[M+Na]⁺.

Compound 8.

S5 (20 mg, 0.044 mmol) and p-toluenesulfonic acid monohydrate (1.68 mg,0.009 mmol) were dissolved in THF (0.5 mL) and methanol (0.5 mL), andstirred under room temperature. After the reaction is complete (2 h),the reaction mixture was diluted with ethyl ether, washed with saturatedaqueous NaHCO₃ solution, and dried with MgSO₄. The solvent was removedunder vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 4:1) to give 8 (16 mg, 74%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 5.35 (dt, J=5.2, 2.0 Hz, 1H), 3.53 (tt,J=11.2, 4.4 Hz, 1H), 2.33-2.23 (m, 4H), 2.01-1.95 (m, 2H), 1.86-1.69 (m,5H), 1.63-1.41 (m, 10H), 1.29-1.05 (m, 7H), 1.00 (s, 3H), 0.94 (d, J=6.4Hz, 3H), 0.68 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 140.9, 121.8, 120.0,71.9, 56.9, 55.9, 50.2, 42.5, 42.4, 39.9, 37.4, 36.6, 35.4, 35.2, 32.0,31.8, 28.4, 24.4, 22.4, 21.2, 19.5, 18.7, 17.7, 12.0; IR (film): 2939,2890, 2345, 2316, 1465, 1063, 960 cm⁻¹; MS (ESI): m/z 368.2 [M-H]⁻.

Compound 5.

Compound 4 (53 mg, 0.121 mmol) and p-toluenesulfonic acid monohydrate(4.6 mg, 0.024 mmol) were dissolved in THF (1 mL) and methanol (1 mL),and stirred under room temperature. After the reaction is complete (2h), the reaction mixture was diluted with ethyl ether, washed withsaturated aqueous NaHCO₃ solution, and dried with MgSO₄. The solvent wasremoved under vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 10:1 to 4:1) to give 5 (36 mg, 84%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 5.34 (dd, J=4.4, 2.0 Hz, 1H), 3.51 (tt,J=11.6, 4.0 Hz, 1H), 2.31-2.05 (m, 4H), 2.01-1.91 (m, 3H), 1.87-1.80 (m,3H), 1.73-1.66 (m, 2H), 1.60-1.40 (m, 8H), 1.33-1.07 (m, 7H), 1.00 (s,3H), 0.92 (d, J=6.8 Hz, 3H), 0.68 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ140.9, 121.8, 85.3, 71.9, 68.0, 56.9, 56.0, 50.2, 42.6, 42.4, 39.9,37.4, 36.6, 35.3, 34.9, 32.0, 31.8, 28.3, 24.4, 21.2, 19.5, 18.3, 15.6,12.0; IR (film): 3302, 2935, 2852, 2334, 1465, 1377, 1135, 1051, 801 cm;MS (ESI): m/z 353.2 [M-H]⁻.

Compound S6.

The mixture of PdCl₂(PPh₃)₂(2.2 mg, 0.003 mmol), CuI (0.6 mg, 0.003mmol), and iodobenzene (7.2 μL, 0.064 mmol) in triethylamine (0.2 mL)was bubbled with Argon gas for ten minutes. To the mixture,triethylamine solution (0.2 mL) of 4 (27.6 mg, 0.063 mmol) was added atroom temperature. After stirring for 5 h at room temperature, thereaction was quenched with saturated NH₄Cl aqueous solution andextracted with ethyl acetate. The organic layer was washed with brine,dried over MgSO₄. The solvent was removed under vacuum, and the residuewas purified by chromatography (Hexane/EtOAc, 40:1) to give S6 (26 mg,79%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 7.38 (d, J=5.2 Hz, 2H), 7.27-7.26 (m, 3H),5.35 (m, 1H), 4.72 (s, 1H), 3.92 (s, 1H), 3.50 (t, J=14.4 Hz, 2H),2.49-2.17 (m, 4H), 2.03-1.71 (m, 9H), 1.56-1.44 (m, 9H), 1.34-1.04 (m,9H), 1.01 (s, 3H), 0.98 (d, J=6.8 Hz, 3H), 0.7 (s, 3H); ¹³C NMR (100MHz, CDCl₃): δ 141.2, 141.1, 131.6, 128.3, 127.6, 124.3, 121.7, 97.1,97.0, 91.0, 80.5, 76.1, 63.1, 63.0, 56.9, 56.0, 50.3, 42.6, 40.4, 39.9,38.9, 37.6, 37.4, 36.9, 35.5, 35.2, 32.1, 31.4, 29.8, 28.4, 28.1, 25.6,24.4, 21.2, 20.3, 20.2, 19.5, 18.5, 16.7, 12.0; IR (film): 2942, 2864,2333, 1492, 1201, 1136, 1024, 972 cm⁻¹; MS (ESI): m/z 537.4 [M+Na]⁺.

Compound 6.

S6 (26 mg, 0.05 mmol) and p-toluenesulfonic acid monohydrate (9.5 mg,0.05 mmol) were dissolved in THF (1 mL) and methanol (1 mL), and stirredunder room temperature. After the reaction was complete, the reactionmixture was diluted with ethyl ether, washed with saturated aqueousNaHCO₃ solution, and dried with MgSO₄. The solvent was removed undervacuum, and the residue was purified by chromatography (Hexane/EtOAc,4:1) to give 6 (21 mg, 98%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 7.38 (d, J=5.6 Hz, 2H), 7.27-7.26 (m, 3H),5.35 (s, 1H), 3.55-3.50 (m, 1H), 2.49-2.20 (m, 4H), 2.04-1.74 (m, 7H),1.58-1.45 (m, 8H), 1.34-1.08 (m, 7H), 1.01 (s, 3H), 0.98 (d, J=6.4 Hz,3H), 0.70 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 140.9, 131.6, 128.3,127.6, 124.3, 121.8, 91.0, 80.5, 71.9, 56.9, 56.0, 50.2, 42.6, 42.4,39.9, 37.4, 36.6, 35.5, 35.2, 32.0, 31.8, 28.3, 24.4, 21.2, 19.5, 18.5,16.7, 12.0; IR (film): 2934, 2851, 2325, 1597, 1466, 1376, 1132, 1108,1040 cm⁻¹; MS (ESI): m/z 453.2 [M+Na]⁺.

Compound S7.

A vial was charged with CuI (1.7 mg, 0.009 mmol), P(o-Tol)₃ (5.5 mg,0.018 mmol), K₂CO₃ (24.9 mg, 0.18 mmol), 4 (40 mg, 0.09 mmol), compoundA (freshly synthesized according to the literature route,³ 21.2 mg,0.117 mmol), and anhydrous EtOH (3 mL). The mixture was stirred for 12 hat 100° C. then cooled to room temperature. The mixture was diluted withEtOAc, filtered through a pad of Celite, and concentrated under vacuo.The residue was purified by chromatography (Hexane/EtOAc, 20:1) to giveS7 (25 mg, 51%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 7.47 (dd, J=4.0, 2.0 Hz, 2H), 7.35-7.27 (m,3H), 5.35 (t, J=6.0 Hz, 1H), 4.72 (t, J=4.0 Hz, 1H), 3.56-3.45 (m, 2H),2.37-2.23 (m, 4H), 2.02-1.94 (m, 2H), 1.86-1.83 (m, 5H), 1.75-1.71 (m,2H), 1.59-1.44 (m, 10H), 1.32-1.08 (m, 8H), 1.01 (s, 3H), 0.94 (d, J=6.8Hz, 3H), 0.70 (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ 141.2, 141.0, 132.6,128.9, 128.4, 122.3, 121.6, 121.6, 97.1, 97.0, 85.4, 76.2, 74.8, 74.6,65.0, 63.0, 63.0, 56.9, 56.0, 50.3, 50.3, 42.6, 40.4, 39.9, 38.9, 37.6,37.4, 36.9, 36.9, 35.4, 34.7, 32.0, 31.4, 29.8, 28.3, 28.1, 25.6, 24.4,21.2, 20.2, 20.2, 19.5, 18.3, 16.8, 12.0; IR (film): 2938, 2868, 2325,1508, 1456, 1116, 1026, 755 cm⁻¹; MS (ESI): m/z 537.3 [M-H]⁻.

Compound 7.

S7 (25 mg, 0.046 mmol) and p-toluenesulfonic acid monohydrate (1.70 mg,0.010 mmol) were dissolved in THF (1 mL) and methanol (1 mL), andstirred under room temperature. After the reaction was complete, thereaction mixture was diluted with ethyl ether, washed with saturatedaqueous NaHCO₃ solution, and dried with MgSO₄. The solvent was removedunder vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 4:1) to give 7 (20 mg, 95%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 7.47 (dd, J=4.0, 2.0 Hz, 2H), 7.33-7.29 (m,3H), 5.36-5.34 (m, 1H), 3.52 (tt, J=11.2, 4.8 Hz, 1H), 2.44-2.20 (m,4H), 2.02-1.95 (m, 2H), 1.85-1.71 (m, 4H), 1.59-1.41 (m, 8H), 1.32-1.08(m, 8H), 1.01 (s, 3H), 0.94 (d, J=6.4 Hz, 3H), 0.70 (s, 3H); ¹³C NMR(100 MHz, CDCl₃): δ 141.0, 132.6, 128.9, 128.5, 122.3, 121.8, 85.4,74.8, 74.6, 71.9, 65.0, 56.9, 56.0, 50.2, 42.6, 42.4, 39.9, 37.4, 36.6,35.5, 34.7, 32.0, 31.8, 28.3, 24.4, 21.2, 19.5, 18.3, 16.8, 12.0; IR(film): 2925, 2853, 2343, 1465, 1377, 1108, 1059 cm⁻¹; MS (ESI): m/z 478[M+Na]⁺.

Compound 9.

S8 (18 mg, 0.033 mmol) and p-toluenesulfonic acid monohydrate (6.3 mg,0.033 mmol) were dissolved in THF (0.7 mL) and methanol (0.7 mL), andstirred under room temperature. After the reaction is complete, thereaction mixture was diluted with ethyl ether, washed with saturatedaqueous NaHCO₃ solution, and dried with MgSO₄. The solvent was removedunder vacuum, and the residue was purified by chromatography(Hexane/EtOAc, 4:1) to give 8 (14 mg, 91%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 7.50 (d, J=8.0 Hz, 2H), 7.37-7.25 (m, 3H),5.35 (d, J=4.0 Hz, 1H), 3.55-3.50 (m, 1H), 2.36-2.24 (m, 4H), 2.01-1.98(m, 2H), 1.85-1.41 (m, 14H), 1.30-1.03 (m, 6H), 1.01 (s, 3H), 0.93 (d,J=6.4 Hz, 3H), 0.69 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 140.9, 133.1,129.6, 128.6, 121.8, 121.3, 83.2, 75.5, 74.8, 71.9, 67.6, 65.6, 59.6,56.8, 55.9, 50.2, 42.6, 42.4, 39.9, 37.4, 36.6, 35.4, 34.4, 32.0, 31.8,28.3, 24.4, 21.2, 19.5, 18.3, 16.8, 12.0; IR (film): 2932, 2335, 2221,1488, 1442, 1376, 1052 cm; MS (ESI): m/z 477.2 [M-H]⁻.

Solubilization of Tagged Cholesterol.

To solubilize the tagged cholesterol molecules, the following procedurewas used to prepare a stock solution of 10 mM cholesterol probemolecules. The appropriate amount of cholesterol probe powder wasdissolved in 100% ethanol to make a 20 mM solution. The tube wasvortexed and then sonicated in bath sonicator for 2 min. The same volumeof DMSO was added into the tube, vortexed, and then sonicated in bathsonicator for 2 min. BODIPY-cholesterol was prepared with the sameprocedure. For SRS imaging and Raman spectral analysis of taggedcholesterol solutions, cholesterol probe molecules were prepared incyclohexanone at 50 mM. The tube was vortexed and then sonicated in bathsonicator for 2 min. 1 aL of the solution was taken to prepare coverglass samples immediately before use.

Cell Culture and PhDY-Chol Treatment.

CHO-K1 cells and M12 cells (mutant CHO-K1 cells that contain a deletionof the NPC1 locus³⁹) were kindly provided by Dr. Daniel Ory, and weregrown in a monolayer at 37° C. in 5% CO₂ in DMEM/F-12 mediumsupplemented with 10% (vol/vol) FBS. To incubate cells with PhDY-Chol,cells were pre-incubated in DMEM/F-12 medium supplemented with 4.4%lipoprotein-deficient serum to deplete medium cholesterol for 16 h. Thecells were then incubated with PhDY-Chol containing medium(DMEM/F-12+4.4% lipoprotein-deficient serum+50 μM PhDY-Chol) for 16 h to24 h. Cells were rinsed with 1×PBS buffer three times before the nextprocedure.

Raman Spectromicroscopy.

The background of Raman spectrum was removed as described. Each Ramanspectrum of tagged cholesterol solution was acquired in 10 seconds, andeach Raman spectrum of fluorescence stained cells was acquired in 30seconds. On the same microscope, TPEF imaging was performed with 707 nmlaser with 100 mW power. Backward-detected two-photon fluorescencesignal was collected through a 425/40 nm or 522/40 nm band-pass filterfor imaging filipin or BODIPY fluorescence, respectively.

Srs Microscopy.

SRS imaging was performed on a femtosecond SRL microscope, with thelaser beating frequency tuned to the C≡C vibration band at 2,252 cm⁻¹,or to the C—H vibration band at 2,885 cm⁻¹, as described previously. Thelaser power at the specimen was maintained at 75 mW, and no cell ortissue damage was observed. For off-resonance, 2,099 cm⁻¹ was used. Onthe same microscope, TPEF imaging was performed with 843 nm laser with30 mW power. Forward-detected two-photon fluorescence signal wascollected through an appropriate band-pass filter for imaging filipin,BODIPY, or LysoTracker.

Acat-1 Inhibition.

ACAT-1 inhibition was used to block cholesterol esterification either byadding a potent ACAT inhibitor, avasimibe, or by RNA interference withACAT-1 shRNA plasmid. Avasimibe: Cells were pre-treated with avasimibeat a final concentration of 10 μM for 24 h. Then PhDY-Chol containingmedium with 10 μM avasimibe was added into the cells and incubated for24 h. RNA interference: RNA interference was employed to specificallyinhibit endogenous ACAT-1. The ACAT-1 shRNA plasmid was purchased fromSanta Cruz (sc-29624-SH). shRNA plasmid was transfected withLipofectamine®2000 (Invitrogen 11668030) as described in themanufacturer's protocols.

HpβCd Treatment.

HPβCD was used as a drug treatment of NP-C disease. M12 cells wereincubated with PhDY-Chol for 16 h as described above. Then cells weretreated with 500 μM HPβCD for 30 h.

Cell-Viability Assay.

CHO cells were grown in 96-well plates with density of 4000 cells perwell. The next day, the cells were treated with each cholesterol probeat the indicated concentrations for 48 h. Cell-viability was measuredwith the MTT colorimetric assay (Sigma).

Propidium Iodide Staining.

Propidium iodide was used to stain late apoptotic or necrotic cells. CHOcells were incubated with 30 μM of tagged cholesterol molecules for 24h. The propidium iodide staining was performed following protocolsprovided by the manufacturer (Life Technologies).

Fluorescent Staining of Free Cholesterol, LDs, and Lysosomes.

Filipin was used to stain free cholesterol. Cells were fixed with 10%formalin solution (Sigma) for 1 h at room temperature. 1.5 mg/mL glycinein PBS was used to quench the formalin by incubating the fixed cells for10 minutes at room temperature. To stain the cells with filipin, workingsolution of 0.05 mg/mL of filipin in PBS/10% FBS was used to incubatecells for 2 h at room temperature. BODIPY was used to label LDs. Cellswere incubated with 10 ag/mL of BODIPY for 30 minutes at roomtemperature. LysoTracker Yellow-HCK-123 was used to stain lysosomesfollowing protocols provided by the manufacturer (Life Technologies).Cells were rinsed with PBS three times before TPEF imaging.

C. elegans Strains.

The N2 Bristol was used as wild-type strain. VC452 strain withchup-1(gk245) X genotype was used to study PhDY-Chol uptake. VS 17strain with hjIs9 [ges-1p::glo-1::GFP+unc-119(+)] genotype was used tostudy cholesterol storage in LROs.

PhDY-Chol Uptake into C. elegans.

PhDY-Chol uptake procedure was modified from a previously reportedprocedures. Briefly, 500 μM of PhDY-Chol in DMSO was spread on the NGMplates seeded with an E. coli OP50 lawn and allowed to grow overnight atroom temperature. C. elegans was then transferred to PhDY-Cholcontaining plates and grown for 3 days before SRS imaging.

Statistical Analysis.

To quantify PhDY-rich area, we first selected one cell and used“Threshold” function to select PhDY-rich cellular regions using ImageJ.Then, by using “Analyze Particles” function, the area fraction (%) ofPhDY-rich region was obtained. To quantify PhDY-rich LDs, “ImageCalculator” function in ImageJ was used to multiply SRS image ofPhDY-Chol by TPEF image of BODIPY. Then, after using “Threshold”function to select PhDY-rich LDs, the number of PhDY-rich LDs wascounted by “Analyze Particles” function. For each group, 7 cells wereanalyzed, and results were shown as mean±standard deviation (SD).Student's t test was used for all the comparisons. p<0.05 was consideredstatistically significant.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

While the inventions have been illustrated and described in detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly certain embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected.

What is claimed is:
 1. A method of forming a probe, the methodcomprising: converting cholenic acid into a compound with a terminalalkyne group, wherein the converting the cholenic acid comprises using asequence, wherein the sequence comprises synthesizing a THP-protectiongroup, LiAlH4 reduction, Dess-Martin oxidation, andSeyferth-Gilbert-Bestmann homologation, wherein the converting thecholenic acid into the compound with the terminal alkyne groupcomprises: adding 3,4-dihydro-2H-pyran (DHP) and p-toluenesulfonic acidmonohydrate (p-TsOH) to a mixture of the cholenic acid and THF;extracting a first residue; adding the first residue to a suspension ofLiAlH₄ in dry THF; extracting a second residue; adding Dess-MartinPeriodinane to a suspension of the second residue and NaHCO₃; extractinga third residue; adding dimethyl-1-diazo-2-oxopropylphosphonate to asolution of the third residue and K₂CO₃; extracting a fourth residue;mixing the fourth residue, CuI, P(o-Tol)₃, K₂CO₃, compound A, andanhydrous EtOH extracting an eleventh residue, wherein the eleventhresidue comprises:


2. The method of claim 1, wherein the residue comprises:


3. The method of claim 1, wherein the second residue comprises:


4. The method of claim 1, wherein the third residue comprises:


5. The method of claim 1, wherein the fourth residue comprises:


6. The method of claim 1, wherein the compound A comprises:


7. The method of claim 1 further comprising: forming alkyne cholesterol(A-Chol) by removing the THP-protection group; forming phenyl-alkynecholesterol (PhA-Chol) from the compound with the terminal alkyne groupvia a palladiumcatalyzed Sonogashira reaction; and forming phenyl-diynecholesterol (PhDY-Chol) from the compound with the terminal alkyne groupvia a coppercatalyzed Cadiot-Chodkiewicz reaction.
 8. The method ofclaim 1, further comprising removing the THP-protection group via anacid.
 9. The method of claim 1 further comprising: addingmethanesulfonyl chloride to a solution of the second residue andtrimethylamine in CH₂Cl₂; extracting a fifth residue, wherein the fifthresidue comprises:


10. The method of claim 9 further comprising: adding potassium cyanideto a solution, wherein the solution comprises the fifth residue andDMSO; extracting a sixth residue, wherein the sixth residue comprises:


11. The method of claim 10 further comprising: dissolving the sixthresidue and p-toluenesulfonic acid monohydrate in THF and methanol;extracting a seventh residue, wherein the seventh residue comprises:


12. The method of claim 1 further comprising: dissolving the fourthresidue and p-toluenesulfonic acid monohydrate in THF and methanol;extracting an eighth residue, wherein the eighth residue comprises:


13. The method of claim 1 further comprising: mixing the fourth residuewith CuI, iodobenzene, and trimethylamine; extracting a ninth residue,wherein the ninth residue comprises:


14. The method of claim 13 further comprising: dissolving the ninthresidue and p-toluenesulfonic acid monohydrate in THF and methanol;extracting a tenth residue, wherein the tenth residue comprises:


15. The method of claim 1 further comprising: mixing the fourth residue,PdCl₂(PPh₃)₂, CuI, compound B, and THF, wherein the compound Bcomprises:

extracting a thirteenth residue, wherein the thirteenth residuecomprises:


16. The method of claim 15 further comprising: dissolving the thirteenthresidue and p-toluenesulfonic acid monohydrate in THF and methanol;extracting a fourteenth residue, wherein the fourteenth residuecomprises:


17. A method of forming a probe, the method comprising: convertingcholenic acid into a compound with a terminal alkyne group, wherein theconverting the cholenic acid comprises using a sequence, wherein thesequence comprises synthesizing a THP-protection group, LiAlH4reduction, Dess-Martin oxidation, and Seyferth-Gilbert-Bestmannhomologation, wherein the converting the cholenic acid into the compoundwith the terminal alkyne group comprises: adding 3,4-dihydro-2H-pyran(DHP) and p-toluenesulfonic acid monohydrate (p-TsOH) to a mixture ofthe cholenic acid and THF; extracting a first residue; adding the firstresidue to a suspension of LiAlH₄ in dry THF; extracting a secondresidue; adding Dess-Martin Periodinane to a suspension of the secondresidue and NaHCO₃; extracting a third residue; addingdimethyl-1-diazo-2-oxopropylphosphonate to a solution of the thirdresidue and K₂CO₃; extracting a fourth residue; mixing the fourthresidue, CuI, P(o-Tol)₃, K₂CO₃, compound A, and anhydrous EtOHextracting an eleventh residue.
 18. The method of claim 17, wherein theeleventh residue comprises:


19. A method of forming a probe, the method comprising: convertingcholenic acid into a compound with a terminal alkyne group, wherein theconverting the cholenic acid comprises using a sequence, wherein thesequence comprises synthesizing a THP-protection group, LiAlH4reduction, Dess-Martin oxidation, and Seyferth-Gilbert-Bestmannhomologation.
 20. The method of claim 19, wherein the converting thecholenic acid into the compound with the terminal alkyne groupcomprises: adding 3,4-dihydro-2H-pyran (DHP) and p-toluenesulfonic acidmonohydrate (p-TsOH) to a mixture of the cholenic acid and THF;extracting a first residue; adding the first residue to a suspension ofLiAlH₄ in dry THF; extracting a second residue; adding Dess-MartinPeriodinane to a suspension of the second residue and NaHCO₃; extractinga third residue; adding dimethyl-1-diazo-2-oxopropylphosphonate to asolution of the third residue and K₂CO₃; extracting a fourth residue;mixing the fourth residue, CuI, P(o-Tol)₃, K₂CO₃, compound A, andanhydrous EtOH extracting an eleventh residue, wherein the eleventhresidue comprises: